Detection Of Protease Inhibitors Research Articles

Gene co-option, duplication and divergence of cement proteins underpin the evolution of bioadhesives across barnacle life histories.

Acquisition of new genes often results in the emergence of novel functions and is a key step in lineage-specific adaptation. As a group of sessile crustaceans, barnacles establish permanent attachment through initial cement secretion at the larval phase followed by continuous cement secretion in juveniles and adults. However, the origins and evolution of barnacle larval and adult cement proteins remain poorly understood. By performing microdissection of larval cement glands, transcriptome and shotgun proteomics and immunohistochemistry validation, we identified 30 larval and 27 adult cement proteins of the epibiotic turtle barnacle Chelonibia testudinaria, of which the majority are stage- and barnacle-specific. While only two proteins, SIPC and CP100K, were expressed in both larvae and adults, detection of protease inhibitors and the cross-linking enzyme lysyl oxidase paralogs in larvae and adult cement. Other barnacle-specific cement proteins such as CP100k and CP52k likely share a common origin dating back at least to the divergence of Rhizocephala and Thoracica. Different CP52k paralogues could be detected in larval and adult cement, suggesting stage-specific cement proteins may arise from duplication followed by changes in expression timing of the duplicates. Interestingly, the biochemical properties of larval- and adult-specific CP52k paralogues exhibited remarkable differences. We conclude that barnacle larval and adult cement systems evolved independently, and both emerged from co-option of existing genes and de novo formation, duplication and functional divergence of lineage-specific cement protein genes. Our findings provide important insights into the evolutionary mechanisms of bioadhesives in sessile marine invertebrates.

Use of fish trypsin immobilized onto magnetic-chitosan composite as a new tool to detect antinutrients in aquafeeds

The unplanned inclusion of antinutrients in fish food affects many biological processes, such as digestibility of amino acids and diet conversion, resulting in undesirable effects on body growth. Thus, the objective of this research was to propose the use of immobilized fish proteases in the detection of protease inhibitors, one of the most important antinutrients. In order to evaluate the detection of antinutritional factors through the immobilized trypsin, the enzyme was incubated with eight diets developed for commercial fish, and residual activity was measured. Comparatively, the tilapia trypsin showed an inhibition of antinutrients (protease inhibitors), present in the eight studied diets, up to 48% greater than the porcine trypsin immobilized in magnetic chitosan. Thus, it is possible to suggest the use of immobilized derivatives containing specific proteases of the target organism in the detection of antinutritional factors that reduce animal’s digestive capacity and negatively influence their growth during husbandry.

Neoleucinodes elegantalis (Lepidoptera: Crambidae): an organism invisible to the defences of tomato fruits

AbstractInsect–plant interactions involving species of the genus Solanum have been intensively studied, resulting in several articles on insect–plant interactions. However, the interactions between herbivores and the fruits of Solanum lycopersicum (tomato) are not well known. Neoleucinodes elegantalis is a borer that causes great yield losses in S. lycopersicum crops because of the direct damage that it causes to the fruits and the difficulty of controlling it. In the field, the outside of a tomato fruit infested with the larvae of N. elegantalis is visually similar to uninfested fruits. Even a minor injury by herbivores can elicit a defensive response. Due to the lack of studies on interactions between fruit borers and S. lycopersicum, our aim in this study was to determine the locations of S. lycopersicum fruit in which the N. elegantalis larvae prefer to feed. An evaluation of nutritional sources was done through histochemical and biochemical tests and the defensive response of the S. lycopersicum fruit to attack by N. elegantalis larvae was evaluated through the detection of protease inhibitors (PIs) and lipoxygenase (LOX) activity. Our results show that the columella region is preferred by the N. elegantalis larvae and that this region has a nutritional source. Furthermore, attack by N. elegantalis larvae in the columella does not induce a significant increase in lipoxygenase activity and PIs. Thus, our results provide a better understanding of the interaction between the larvae of N. elegantalis and S. lycopersicum fruits and a better understanding of the evolution of plant–herbivore interactions, with an emphasis on the choice of feeding location as a strategy to avoid plant defences.

Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane–Tricine buffer system

A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is reported. Inhibitor samples were separated on a protein substrate–SDS–polyacrylamide gel in a Tris–Tricine buffer system that improves the separation and identification of peptides and low-molecular-weight proteins. After electrophoresis, the gel was incubated with the target proteases to hydrolyze the background protein substrate. The inhibitor bands, which were protected from proteolysis by the target proteases, were stained. Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands in the white background when the gels were treated with the target proteases. Some significant applications of this method are introduced. This method is an ideal system for discovering new protease inhibitors in small natural samples.

Detection of protease inhibitors in the hemolymph of resistantAnticarsia gemmatalis which are inhibitory to the entomopathogenic fungus,Nomuraea rileyi

A complex of protease inhibitor activities has been detected in the hemolymph of the 6th instarAnticarsia gemmatalis larvae that are resistant to infection by the fungusNomuraea rileyi. A site-specific serine protease inhibitor extracted fromA. gemmatalis hemolymph inhibits both the germination ofN. rileyi conidia and subsequent germ tube development.